Collagen is often involved in connective tissue disorders. The malfunctioning of any one of the complex series of processes in the biosynthesis of this protein can, potentially, lead to these disorders. The proposed project is concerned with one of the crucial biosynthetic steps which is responsible for the stability of the collagen molecule in its triple-helical ccnformation, namely, the post-translational hydroxylation of its proline residues by proline hydroxylase. Our recent studies have provided an insight into the conformational criteria for the proline hydroxylation; thus, the conformational feature recognized by prolyl hydroxylase appears to be the beta-turns at the Pro-Gly segments of its substrates. What we intend achieving in the proposed studies is the understanding, at the molecular level, of the conformational consequences of this enzymatic process. Our approach to obtaining this information is to mimic the in vivo event by appropriate model systems and observe the conformational changes that take place due to the incorporation of the hydroxyproline residues. We intend to isolate the pure enzyme and study its interaction with synthetic polypeptide substrates with specific sequences, viz. (Gly-X-Pro)n, X equals Ala, Leu, Sar and Val. The substrate conformation before and after, as well as during, the enzymatic hydroxylation will be monitored by spectral techniques. The interpretation of these data will be aided by a parallel study of the conformation and conformational transition of synthetic oligo- and polypeptides containing hydroxyproline, viz. Gly-X-Hyp-Gly-X-OH and (Gly-X-Hyp)n. These will also serve to understand the role of Hyp in collagen structure. Similar studies on Hyp-containing analogs of tropoelastin and bradykinin will also be carried out. Together, these studies will, hopefully, enable us to understand some of the pathological effects in terms of the conformation of the molecules involved.